1. Field of the Invention
The present invention relates to hybridoma cell lines and to monoclonal antibodies produced thereby that are specific for antigens coded by the HLA gene complex and therefore are useful in the typing of human tissue that is to be used in organ transplants.
2. Description of the Prior Art
Transplantation of human organs, particularly the kidney, has become a relatively common procedure. Ideally, the donor organ is obtained from an identical twin since the antigens of the donor and recipient in such a case are identical and no histoincompatibility exists. Therefore, no immune response to the graft occurs in such a transfer, known as an isograft. However, most transplants are between two less closely related individuals of the same species and histocompatibility differences in such an allograft may be stong or weak, depending on the individuals. The fate of transplanted tissues and organs depends on a number of factors, but the recipient's immune response to graft antigens is the central event. Definition of antigenic systems which serve as strong barriers to transplantation has therefore become a major investigational interest, having both practical application in clinical transplantation and theoretical value in understanding the natural role of the histocompatibility antigens in immunobiology.
A single chromosomal gene complex codes for the major histocompatibility antigens in each vertebrate species investigated so far. In humans the histocompatibility antigens are produced by the HLA gene complex. This complex occupies a portion of the short arm of the human C6 chromosome and consists of several series of paired alleles which are inherited from generation to generation in a dominant fashion, segregating randomly from other important antigens such as the ABH red blood cell type groups.
Antigens of the HLA system are divided into two classes. Each class I antigen consists of an 11,500-dalton .beta..sub.2 -microglobulin sub-unit and a 44,000-dalton heavy chain which carries the antigenic specificity. Three gene loci (A, B and C) are recognized for the class I antigens. There are over 60 clearly defined A and B specificities while 8 C locus specificities are known. Evidence that this gene complex plays the major role in the transplantation response comes from the fact that haplotype-matched sibling donor-recipient combinations show excellent results in kidney transplantation, in the vicinity of 85% to 90% long term survival.
A non-serologically defined antigen, responsible for the mixed lymphocyte response, is caused by a distinct locus called D. Although D-locus antigens are not as yet clearly identifiable by serotyping techniques, serologically defined specificities closely related to the D-locus have been defined. These have the special property of not being expressed on platelets or unstimulated T lymphocytes. These specificities are termed class II, having two glycoprotein chains of 29,000 (.beta.) and 34,000 (.alpha.) daltons and lacking .beta..sub.2 globulin. These antigens are also termed HLA-DR (D-related) and are important in tissue typing.
Tissue typing is currently being carried out using sera obtained from multiparous women. A major problem exists because of the unreliability of this source. Only a limited amount of antibody is available from any one woman. Accordingly, it is necessary to continually replace standard antibodies with new antibodies which must be standardized and checked against the previously existing ones. Furthermore, because of the heterogeneous nature of antibodies obtained in this fashion, cross-reactivity is a major problem. Accordingly, there have been many attempts to produce antibodies more suitable for crossmatching by immunological techniques. Several such attempts involve xenoimmune (cross-species) sera to detect allospecificity in the antigen-donor species. Examples include rabbit anti-human antibodies by Einstein et al, Transplantation 12:299 (1971) and Ferrone, Tissue Antigens 12:153 (1978) and monkey anti-human antibodies by Sanderson et al, Transplant. Proc. 2:163 (1974). The most specific anti-HLA serum appears to be a rabbit anti-A9 serum prepared by immunization with A9 antigen purified from human serum or urine (see Ferrone et al, Tissue Antigens 5:41 (1975)). However, A9 is actually a common determinate of A23 and A24, and thus possibly the allele-specific major epitope is not the only target with which this antibody reacts.
The advent of hydridomal techniques has brought about the possibility of producing homogeneous populations of highly specific antibodies gainst a variety of antigens. Koprowski et al, U.S. Pat. No. 4,172,124, describes antibodies produced by somatic cell hybrids between myeloma cells and spleen or lymph cells that are specific for malignant tumors. Koprowski et al, U.S. Pat. No. 4,196,265, describes continuous cell lines of genetically-stable fused-cell hybrids capable of producing large amounts of IgG antibodies against specific viruses, such as influenza virus, rabies, mumps, SV40 and the like. Zurawski et al, Federation Proceedings 39: 4922 (1980), discloses hybridomas producing monoclonal IgG antibodies against tetanus toxin. Monoclonal antibodies have also been described against human tumor cells, Yeh et al, Proc. Nat. Acad. Sci. 76: 2927 (1979); human T lymphocyte subsets, Reinberz et al, Proc. Nat. Acad. Sci. 76: 4062 (1979) and malaria parasites, Nussenzweig et al, Science 207: 71 (1980).
The prior art most closely related to this application appears to be McKearn et al, "Rat-Mouse Hybridomas and Their Application to Studies of the Major Histocompatibility Complex," in Monoclonal Antibodies, Kennett et al, eds. Plenum Publishing Co., N.Y. (1981), which discloses the use of rat-mouse hybridomas and their application to studies of the histocompatibility complex in various species. Several rat-mouse hybridomas producing rat anti-rat antibodies were reactive with determinants on cells from other species, such as humans. Four of nine hybridoma antibodies were reactive with periphrial blood lymphocytes from randomly chosen humans in a complement-mediated cytotoxicity assay. However, a later study by the same investigators indicated that these antibo dies were not useful in identifying private determinants of the HLA locus since no correlation between specific HLA antigens and the hybridomally produced antibodies occurred.
Accordingly, there still exists a need for monoclonal antibodies suitable for tissue matching.